Polypeptide

ABSTRACT

The present invention provides 5T4 tumour-associated antigen (TAA) for use in a method of immunotherapy of tumours. The invention also relates to a recombinant poxvirus vector from which at least one immune evasion gene has been deleted, which comprises a nucleic acid sequence encoding a 5T4 TAA and the use thereof in vaccinating against and in treating tumours.

This application claims the benefit of Provisional application60/126,187 filed Mar. 25, 1999 and Provisional application 60/126,188filed Mar. 25, 1999.

FIELD OF THE INVENTION

The present invention relates to a tumour-associated antigen (TAA)useful for eliciting an anti-tumour immunotherapeutic response insubjects. In particular, the invention relates to 5T4 antigen and itsuse in immunotherapy.

BACKGROUND TO THE INVENTION

A number of oncofoetal or tumour-associated antigens (TAAs) have beenidentified and characterised in human and animal tumours. In general,TAAs are antigens expressed during foetal development which aredownregulated in adult cells, and are thus normally absent or presentonly at very low levels in adults. Tumour cells have been observed toresume expression of TAAs, and the application of TAAs for tumourdiagnosis, targeting and immunotherapy has therefore been suggested.

In particular, the recent cloning of tumour antigens recognised by Tcells has caused considerable interest in the development of antigenspecific cancer vaccines. However, many tumour associated antigens arenon-mutated, poorly immunogenic tissue differentiation antigens. Theirweak immunogenicity may be due to self tolerance. Thus they are rarelyindicated as antigenic peptides suitable for raising an immune response.

Notwithstanding this, some tumour associated antigens are found to beregularly associated with tumours in a large number of individuals. Suchantigens are especially attractive candidates for use in vaccines. Theyinclude the melanoma differentiation antigens (MDA), melanoma antigenswhich are recognised by T lymphocytes as well as several proteins in theMAGE family. However, as indicated by results from clinical trialsobtained to date, inducing therapeutic T cells to these antigens hasproved extremely difficult. One reason for the apparenthyporesponsiveness of the human immune system to many tumour antigensmay be that they are normal, non-mutated self antigens, expressed onnormal tissues as well as on tumour cells. The immune system is not ableto differentiate the tumour antigen on a tumour cell from ordinary, selfproteins.

A major barrier to the application of tumour immunotherapy approachesusing non-mutated self cellular antigens is thus apparently the breakingof tolerance to such an antigen. For example, a murine zona pellucidaantigen expressed by a murine poxvirus recombinant was able to induceinfertility in mice. These data indicate that though the breaking oftolerance using recombinant pox viruses expressing self antigens ispossible, there is still a requirement to optimise their efficacy suchthat the active treatment of established tumours becomes possible.

The TAA 5T4 (see WO 89/07947) has been extensively characterised. It isa 72 kDa glycoprotein expressed widely in carcinomas, but having ahighly restricted expression pattern in normal adult tissues (see Table1). It appears to be strongly correlated to metastasis in colorectal andgastric cancer. The full nucleic acid sequence of human 5T4 is known(Myers et al., 1994 J Biol Chem 169: 9319–24).

TABLE 1 Distribution of Human 5T4 Tumour 5T4 Frequency Type (%) Breast84 Ovarian 71 Gastric 74 Colorectal 85(Starzynska et al., Eur J Gastroenterol Hepatol 1998 June;10(6):479–84;Starzynska et al., Br J Cancer 1994 May;69(5):899–902; Starzynska etal., Br J Cancer 1992 November;66(5):867–9)

Although 5T4 has been proposed as a marker, with possible mechanisticinvolvement, for tumour progression and metastasis potential (Carsberget al., (1996) Int J Cancer 1996 Sep. 27;68(1):84–92), 5T4 has not beenproposed for use as an immunotherapeutic agent. The breaking of immunetolerance to 5T4, which is itself expressed in a restricted manner inadult tissues, has not been demonstrated. Thus, it could not bepredicted whether 5T4 could prove to be an effective antigen forimmunotherapy against cancer.

SUMMARY OF THE INVENTION

If a successful therapeutic outcome is to be achieved, animmunotherapeutic approach to cancer treatment depends on a number offactors. These include the ability to elicit a cytotoxic T-lymphocyte(CTL) response, the ability to elicit an antibody response and,importantly, the ability to break immune tolerance in a subject. It hasnow been demonstrated that immunisation of subjects with 5T4 results ina successful immunotherapeutic response as judged by the above. Inparticular, immunisation with 5T4 has been shown to elicit an antibodyresponse.

Accordingly, the present invention provides a viral vector expressing anucleic acid encoding 5T4 antigen.

Expression of 5T4 antigen in a subject is effective in eliciting animmunotherapeutic anti-tumour response. Preferably, the viral vectorfavours CTL responses to expressed antigens, and is advantageously apoxvirus vector, such as a vaccinia virus vector. Further vectors, bothviral and non-viral, which are suitable for delivering 5T4 antigen aredescribed below.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors,plasmids, naked nucleic acids, nucleic acids complexed with polypeptideor other molecules and nucleic acids immobilised onto solid phaseparticles. Such vectors are described in detail below. It will beunderstood that the present invention, in its broadest form, is notlimited to any specific vector for delivery of the 5T4-encoding nucleicacid.

A “nucleic acid”, as referred to herein, may be DNA or RNA,naturally-occurring or synthetic, or any combination thereof. Nucleicacids according to the invention are limited only in that they serve thefunction of encoding 5T4 antigen in such a way that it may be translatedby the machinery of the cells of a host organism. Thus, natural nucleicacids may be modified, for example to increase the stability thereof.DNA and/or RNA, but especially RNA, may be modified in order to improvenuclease resistance of the members. For example, known modifications forribonucleotides include 2′-O-methyl, 2′-fluoro, 2′-NH₂, and 2′-O-allyl.The modified nucleic acids according to the invention may comprisechemical modifications which have been made in order to increase the invivo stability of the nucleic acid, enhance or mediate the deliverythereof, or reduce the clearance rate from the body. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions of a given RNA sequence. See, forexample, WO 92/03568; U.S. Pat. No. 5,118,672; Hobbs et al., (1973)Biochemistry 12:5138; Guschlbauer et al., (1977) Nucleic Acids Res.4:1933; Schibaharu et al., (1987) Nucleic Acids Res. 15:4403; Pieken etal., (1991) Science 253:314, each of which is specifically incorporatedherein by reference.

5T4 antigen is “expressed” in accordance with the present invention bybeing produced in the cells of a host organism as a result oftranslation, and optionally transcription, of the nucleic acid encoding5T4. Thus, 5T4 is produced in situ in the cell. Since 5T4 is atransmembrane protein, the extracellular portion thereof is displayed onthe surface of the cell in which it is produced. If necessary,therefore, the term “expression” includes the provision of the necessarysignals to ensure correct processing of 5T4 such that it is displayed onthe cell surface and can interact with the host immune system.

As used herein, the term “polypeptide” refers to a polymer in which themonomers are amino acids and are joined together through peptide ordisulphide bonds.

“Polypeptide” refers to a full-length naturally-occurring amino acidchain or a fragment thereof, such as a selected region of thepolypeptide that is of interest in a binding interaction, or a syntheticamino acid chain, or a combination thereof. “Fragment thereof” thusrefers to an amino acid sequence that is a portion of a full-lengthpolypeptide, between about 8 and about 500 amino acids in length,preferably about 8 to about 300, more preferably about 8 to about 200amino acids, and even more preferably about 10 to about 50 or 100 aminoacids in length. Additionally, amino acids other thannaturally-occurring amino acids, for example β-alanine, phenyl glycineand homoarginine, may be included. Commonly-encountered amino acidswhich are not gene-encoded may also be used in the present invention.

5T4 antigen is the polypeptide known as 5T4 and characterised, forexample, in WO89/07947. In a preferred aspect, 5T4 is human 5T4 ascharacterised by Myers et al ibid., the sequence of which appears inGenBank at accession no. Z29083 and is set out herein as SEQ. ID. No. 1.The invention however comprises species and allelic variations of 5T4,including canine 5T4 set forth herein at SEQ. ID. No. 3 and mouse 5T4set forth herein at SEQ. ID. No. 2 (GenBank Accession no. AJ012160), aswell as fragments, preferably distinct epitopes, and variants thereofcomprising amino acid insertions, deletions or substitutions whichretain the antigenicity of 5T4. Such fragments and variants aredescribed in greater detail below.

In a second aspect, the present invention relates to a modified 5T4antigen. A “modified” antigen, as used herein, is a 5T4 polypeptidewhich has been truncated, extended or otherwise mutated such that itdiffers from naturally-occurring 5T4. It has been found that peptidefragments derived from 5T4 are able to function as 5T4-specificantigenic determinants. Such peptides are able to bind HLA molecules andto induce CTL responses against wild-type 5T4 in subjects, often moreeffectively that full-length 5T4. Moreover, 5T4 peptides may be mutated,by amino acid insertion, deletion or substitution; mutated peptidesadvantageously bind even more effectively to HLA and elicit an even morepotent CTL response in subjects. Peptides may be any length, but areadvantageously between 5 and 25 amino acids, preferably between 6 and 15amino acids, and advantageously about 9 amino acids in length.

Modified peptides are advantageously HLA CTL epitopes of 5T4.Modification of such epitopes may be performed based on predictions formore efficient CTL induction derived using the program “Peptide BindingPredictions” devised by K. Parker (NIH) which may be found athttp://www-bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform (seealso Parker, K. C et al. 1994.J.Immunol. 152:163).

In a preferred aspect, a “modified” 5T4 peptide includes peptides whichhave been bound or otherwise associated to transporter peptides oradjuvants, in order to increase their ability to elicit an immuneresponse. For example, peptides may be fused to TAP independenttransporter peptides for efficient transport to HLA and interaction withHLA molecules to enhance CTL epitopes (for review see Yewdell et al.,1998 J Immunother 21:127–31; Fu et al., (1998) J Virol 72:1469–81).

In a third aspect, the present invention provides a method for elicitingan immune response in a subject, comprising the steps of immunising thesubject with a nucleic acid encoding 5T4 antigen, and expressing the 5T4antigen in the subject.

An immune response is elicited, as stated, by immunisation with5T4-expressing nucleic acid. Immunisation may be elicited through theadministration of a “priming” agent comprising an antigen followed by asecondary or “boosting” agent comprising additional antigen which isadministered to the immune system after it has been efficiently primedwith the priming agent.

The vector employed for immunisation may be any vector, viral ornon-viral. The 5T4 antigen used, whether full length 5T4 or peptidesthereof, may be modified and may be homologous (i.e. derived from thesame species as the subject) or heterologous in origin.

Preferably, the immune response elicited is a CTL response whichinvolved the activation of cytotoxic T-lymphocytes which are 5T4specific.

Advantageously, the response is an anti-tumour immunotherapeuticresponse which is effective to inhibit, arrest or reverse thedevelopment of a tumour in a subject.

In a fourth aspect, the present invention provides the use of a 5T4antigen in the preparation of a composition for the immunotherapy of atumour in a subject.

Advantageously, immunisation with a 5T4 antigen is capable of breakingimmune tolerance to 5T4 in a subject.

According to a fifth aspect, the present invention provides a vaccinecomposition comprising 5T4 antigen. The vaccine composition may comprisea homologous 5T4 antigen, a heterologous 5T4 antigen or a mutant 5T4antigen.

5T4 antigen-containing vaccines are useful for immunisation against, ortherapy of, tumours, in a manner analogous to the use of 5T4-encodingnucleic acids for the same purposes.

Advantageously, the vaccine composition comprises one or more adjuvants.

In a sixth aspect, the invention comprises an expression vector encodinga 5T4 antigen, which vector is useful for the expression of 5T4 and theproduction of 5T4 antigen suitable for use in a vaccine composition. Thevector may be a prokaryotic or eukaryotic vector, and is advantageouslya vector capable of expressing 5T4 in mammalian cells.

The 5T4 antigen may be from any source, and may be a modified 5T4antigen, for example as set forth herein.

In a seventh aspect, the present invention provides the use of a 5T4antigen in the preparation of a composition for immunising a subject.Immunisation using a 5T4 comprises administering to the subject animmunologically effective amount of the vaccine composition according tothe fifth aspect of the invention.

In an eighth aspect, the present invention provides the use of a 5T4antigen in the preparation of a composition for the sterilisation of asubject. The administration of 5T4 antigen may be effective in causingsterilisation of subjects. Preferably, the subject is a female subject.

The invention further relates to the use of 5T4 targeting molecules,such as anti-5T4 antibodies, for example anti-5T4 scFvs. Theseantibodies may be used to (i) to target natural or exogenous 5T4 in situand/or (ii) deliver immune enhancer molecules, such as B7.1, to naturalor exogenous 5T4 in situ (Carroll et al. (1998) J Natl Cancer Inst90(24):1881–7). This potentiates the immunogenicity of 5T4 in thesubject. The present invention also relates to the sequential use of avector encoding a 5T4 antigen and anti-5T4 antibodies, for example ananti-5T4 scFvs. The anti-5T4 scFvs antibodies may be administered asnaked DNA encoding the antibodies (for example, in a plasmid comprisingthe encoding DNA together with a short promoter region to control itsproduction), in an expression vector (which may be viral or non-viral)comprising the encoding sequence or in a protein form. Thus, theinvention provides a vector encoding a 5T4 antigen and an agent capableof binding 5T4 which is optionally fused with an immunostimulatorymolecule, for separate, such as sequential use, in the treatment oftumours.

In a further embodiment, the invention encompasses a combination therapyincluding enzyme/prodrug therapy and immunotherapy with 5T4. Forexample, the enzyme/prodrug therapy may comprise intratumoural orsystemic delivery of P450, delivered optionally using an retroviral orlentiviral vector, and cyclophosphamide (CPA) followed by systemicimmunotherapeutic induction with 5T4.

Thus, the invention further relates to a vector encoding 5T4 antigen aprodrug/enzyme combination, for separate, simultaneous separate orcombined use in the treatment of tumours.

In a further embodiment, 5T4 or 5T4 peptides may be fused to hepatitis Bcore antigen to enhance T helper and antibody responses (Schodel et al.,1996 Intervirology 39:104–10).

In accordance with a ninth aspect of the invention, therefore, there isprovided a recombinant poxvirus vector from which at least one immuneevasion gene has been deleted, which comprises a nucleic acid sequenceencoding a tumour-associated antigen (TAA).

TAAs are weakly immunogenic, being recognised as “self” by the immunesystem and thus tolerated to a large extent. Although the use ofpoxvirus vectors is able to cause the antigens to be presented such thatthis tolerance may be overcome at least in part, the immunogenic effectobserved with most poxvirus vectors is limited. It is thought that thedeletion of immune evasion genes, naturally present in poxviruses, mayhave a beneficial effect in vaccination with TAAs. Poxvirus vectorshaving deleted immune evasion genes may be capable of breaking immunetolerance to encoded self antigens, including TAAs, thus enabling a hostto raise an immune response to a poorly immunogenic or other selfantigen.

In a tenth aspect, the present invention provides a method for elicitingan immune response in a mammal, comprising administering to the mammal arecombinant poxvirus vector according to the ninth aspect of theinvention, thereby eliciting an immune response to the TAA in themammal.

Antigens such as TAAs are known to rely on the generation of a CTLresponse in order to provide a protective or therapeutic effect in asubject, which is dependent on processing of antigen via the MHCIpathway. Long-term antigen expression is thought to lead to increasedlongevity of high level CTL. In an eleventh aspect of the invention,there is provided a poxvirus having a reduced lytic activity for theenhancement of a CTL response to an antigen in a subject.

In a twelfth aspect, the present invention provides the use of arecombinant poxvirus vector according to the nineth or eleventh aspectof the invention, to elicit an immune response in a mammal against aTAA.

In a thirteenth aspect, the invention provides a 5T4 antigen for use asa tumour-associated target in immunotherapy.

In a fourthteenth aspect, the invention provides the use of arecombinant poxvirus vector from which at least one immune evasion genehas been deleted or mutated, which comprises a nucleic acid sequenceencoding a weak immunogen, to break immune tolerance in a mammal againstthe weak immunogen and elicit an immune response thereto.

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and discussion. These aspectsare presented under separate section headings. However, it is to beunderstood that the teachings under each section heading are notnecessarily limited to that particular section heading.

DETAILED DESCRIPTION OF THE INVENTION

Vectors for Delivery or Expression of 5T4 Antigen

5T4 polypeptides in accordance with the present invention can bedelivered by viral or non-viral techniques.

Non-viral delivery systems include but are not limited to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a 5T4 gene to a target mammalian cell.

Typical transfection methods include electroporation, nucleic acidbiolistics, lipid-mediated transfection, compacted nucleic acid-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), multivalent cations such as spermine, cationic lipids orpolylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 NatureBiotechnology 16: 421) and combinations thereof.

Viral delivery systems include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes viral vectors,retroviral vectors, lentiviral vectors or baculoviral vectors,Venezuelan equine encephalitis virus (VEE), poxviruses such as:canarypox virus (Taylor et al 1995 Vaccine 13:539–549), entomopox virus(Li Y et al 1998 XII^(th) International Poxvirus Symposium p144.Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637–46)alphavirus, and alphavirus based DNA vectors.

Examples of retroviruses include but are not limited to: murineleukaemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV).

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758–763).

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053–3058;Lewis and Emerman 1994 J. Virol. 68: 510–516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

The vector of the present invention may be configured as a split-intronvector. A split intron vector is described in PCT patent applications WO99/15683 and WO 99/15684.

If the features of adenoviruses are combined with the genetic stabilityof retroviruses/lentiviruses then essentially the adenovirus can be usedto transduce target cells to become transient retroviral producer cellsthat could stably infect neighbouring cells. Such retroviral producercells engineered to express 5T4 antigen can be implanted in organismssuch as animals or humans for use in the treatment of angiogenesisand/or cancer.

Poxvirus Vectors

Poxvirus vectors are preferred for use in the present invention. Poxviruses are engineered for recombinant gene expression and for the useas recombinant live vaccines. This entails the use of recombinanttechniques to introduce nucleic acids encoding foreign antigens into thegenome of the pox virus. If the nucleic acid is integrated at a site inthe viral DNA which is non-essential for the life cycle of the virus, itis possible for the newly produced recombinant pox virus to beinfectious, that is to say to infect foreign cells and thus to expressthe integrated DNA sequence. The recombinant pox virus prepared in thisway can be used as live vaccines for the prophylaxis and/or treatment ofpathologic and infectious disease.

Expression of 5T4 in recombinant pox viruses, such as vaccinia viruses,requires the ligation of vaccinia promoters to the nucleic acid encoding5T4. Plasmid vectors (also called insertion vectors), have beenconstructed to insert nucleic acids into vaccinia virus throughhomologous recombination between the viral sequences flanking thenucleic acid in a donor plasmid and homologous sequence present in theparental virus (Mackett et al 1982 PNAS 79: 7415–7419). One type ofinsertion vector is composed of: (a) a vaccinia virus promoter includingthe transcriptional initiation site; (b) several unique restrictionendonuclease cloning sites located downstream from the transcriptionalstart site for insertion of nucleic acid; (c) nonessential vacciniavirus sequences (such as the Thymidine Kinase (TK) gene) flanking thepromoter and cloning sites which direct insertion of the nucleic acidinto the homologous nonessential region of the virus genome; and (d) abacterial origin of replication and antibiotic resistance marker forreplication and selection in E. Coli. Examples of such vectors aredescribed by Mackett (Mackett et al 1984, J. Virol. 49: 857–864).

The isolated plasmid containing the nucleic acid to be inserted istransfected into a cell culture, e.g., chick embryo fibroblasts, alongwith the parental virus, e.g., poxvirus. Recombination betweenhomologous pox DNA in the plasmid and the viral genome respectivelyresults in a recombinant poxvirus modified by the presence of thepromoter-gene construct in its genome, at a site which does not affectvirus viability.

As noted above, the nucleic acid is inserted into a region (insertionregion) in the virus which does not affect virus viability of theresultant recombinant virus. Such regions can be readily identified in avirus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton, et al J. Virology 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox andvariola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour virus);avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988) (fowlpox); Boyle,et al Virology 156:355 (1987) (fowlpox); Schnitzlein, et al J.Virological Method, 20:341 (1988) (fowlpox, quailpox); entomopox(Lytvyn, et al J. Gen. Virol 73:3235–3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, HindIII M.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, BamHI J [Jenkins, et al AIDS Research and HumanRetroviruses 7:991–998 (1991)] the EcoRI-HindIII fragment, BamHIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308 220 A1. [Calvert, et alJ. of Virol 67:3069–3076 (1993); Taylor, et al Vaccine 6:497–503 (1988);Spehner, et al (1990) and Boursnell, et al J. of Gen. Virol 71:621–628(1990)].

In swinepox preferred insertion sites include the thymidine kinase generegion.

A promoter can readily be selected depending on the host and the targetcell type. For example in poxviruses, pox viral promoters should beused, such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificialconstructs containing appropriate pox sequences can also be used.Enhancer elements can also be used in combination to increase the levelof expression. Furthermore, the use of inducible promoters, which arealso well known in the art, are preferred in some embodiments.

Foreign gene expression can be detected by enzymatic or immunologicalassays (for example, immuno-precipitation, radioimmunoassay, orimmunoblotting). Naturally occurring membrane glycoproteins producedfrom recombinant vaccinia infected cells are glycosylated and may betransported to the cell surface. High expressing levels can be obtainedby using strong promoters.

Other requirements for viral vectors for use in vaccines include goodimmunogenicity and safety. MVA is a replication-impaired vaccinia strainwith a good safety record. In most cell types and normal human tissue,MVA does not replicate. Replication of MVA is observed in a fewtransformed cell types such as BHK21 cells. Carroll et al (1997) haveshown that the recombinant MVA is equally as good as conventionalrecombinant vaccinia vectors at generating a protective CD8+T cellresponse and is an efficacious alternative to the more commonly usedreplication competent vaccinia virus. The vaccinia virus strains derivedfrom MVA, or independently developed strains having the features of MVAwhich make MVA particularly suitable for use in a vaccine, are alsosuitable for use in the present invention.

Preferably, the vector is a vaccinia virus vector such as MVA or NYVAC.Most preferred is the vaccinia strain modified virus ankara (MVA) or astrain derived therefrom. Alternatives to vaccinia vectors includeavipox vectors such as fowlpox or canarypox known as ALVAC and strainsderived therefrom which can infect and express recombinant proteins inhuman cells but are unable to replicate.

In one aspect of the present invention at least one immune evasion geneis deleted from the poxvirus vector.

Viruses, especially large viruses such a poxviruses which have anextensive coding capacity and can thus encode a variety of genes, havedeveloped a number of techniques for evading the immune system of theirhosts. For example, they are able to evade non-specific defences such ascomplement, interferons and the inflammatory response, as well as tointerfere with or block the function of cytokines. A number of theseimmune evasion polypeptides have been deleted from MVA, with theexception of the interferon resistance protein in the left terminalregion.

Poxviruses in general, being large DNA viruses which establish acute,rather than latent, infections. They encode so many antigenic proteinsthat antigenic variation is difficult, thus relying on active immuneevasion to protect themselves from the mammalian immune system. Theypossess a number of genes which encode polypeptides which areresponsible for interfering with a number of aspects of the immunesystem: they disrupt interferon action, interfere with complement,cytokine activity, inflammatory responses and CTL recognition (for areview, Smith et al., (1997) Immunol Rev 159:137–154). Removal of theseproteins is beneficial in promoting the ability of weak immunogensencoded on a poxvirus vector to elicit an immune response in a subject.

An immune evasion gene or polypeptide is a gene, or its product, whichassists the virus in evading the mammalian immune system. Preferably,the gene or gene product interferes with the working of the immunesystem, at least one level. This may be achieved in a number of ways,such as by interfering in signalling pathways by providing competitorsfor signalling molecules, by providing soluble cytokine receptor mimicsand the like.

Immune evasion genes include, but are not limited to, the following:

Interferon evasion genes. Vaccinia possesses at least three genes whichinterfere with IFN action. The E3L gene expresses a 25 Kd polypeptidewhich competes with P1 protein kinase for binding to dsRNA, an eventwhich leads to activation of P1, phosphorylation of eIF2α and resultantfailure of translation initiation complex assembly. This pathway isordinarily responsive to IFN activation, but is impeded by E3Lexpression thus allowing translation initiation to proceed unimpeded.

The K3L gene expresses a 10.5 Kd polypeptide which also interferes withP1 activity, since it is effectively an eIF2α mimic and acts as acompetitor for P1 protein kinase. Its mode of action is thus similar toE3L.

The A18R gene is predicted to encode a helicase, which appears tointerfere with the 2′,5′-oligoadenylate pathway, which is in turn IFNresponsive. 2′,5′-A activates RNAse L, which acts to prevent viraltranslation. Expression of A18R appears to reduce 2′,5′-A levels ininfected cells.

Complement. The product of the B5R gene of vaccinia is known to behighly related to factor H, a regulator of the alternative complementpathway. This pathway may be activated by antigen alone, unlike theclassical pathway. The B5R gene product thus may interfere with thealternative complement pathway.

The C21L gene is in turn related to C4b-binding protein in humans, andinteracts with cells bearing C4b on the surface to prevent binding tothe CR1 complement receptor.

Soluble Cytokine Receptors. The product of the vaccinia WR B15R gene(B16R in Copenhagen strain vaccinia) is related to IL1-R, the receptorfor IL-1β.

The WR gene ORF SalF19R, A53R in Copenhagen strain vaccinia, encodes aTNF receptor. However, in wild-type virus both of these genes arebelieved to be inactive due to fragmentation of the ORFs.

The B8R gene is believed to encode a soluble IFN-γ receptor, providingthe virus with yet another IFN evasion mechanism.

Inflammation. A number of genes are believed to be involved in theprevention of inflammatory responses to viral infection. These includeA44L, K2L, B13R and B22R.

In one aspect of the present invention, the majority of the immuneevasion genes are deleted from the recombinant poxvirus vector.Preferably, all the immune evasion genes are deleted. Thus, in oneaspect of the present invention, the recombinant poxvirus vector is arecombinant MVA vector in which the K3L interferon resistance proteingene has been disrupted or deleted.

Preferred are poxviruses which are non-hazardous to the intendedsubject. Thus, for example, for use in humans, poxviruses which areeither host-range restricted, such as avipox viruses, or otherwiseattenuated, such as attenuated strains of vaccinia (including NYVAC andMVA) are preferred. Most preferred are attenuated vaccinia virusstrains, although non-vaccinia strains are usefully employed in subjectswith pre-existing smallpox immunity.

A construct which contains at least one nucleic acid which codes for 5T4flanked by MVA DNA sequences adjacent to a naturally occurring deletion,e.g. deletion II, within the MVA genome, is introduced into cellsinfected with MVA, to allow homologous recombination.

Once the construct has been introduced into the eukaryotic cell and the5T4 DNA has recombined with the viral DNA, the desired recombinantvaccinia virus, can be isolated, preferably with the aid of a marker(Nakano et al Proc. Natl. Acad. Sci. USA 79, 1593–1596 [1982], Franke etal Mol. Cell. Biol. 1918–1924 [1985], Chakrabarti et al Mol. Cell. Biol.3403–3409 [1985], Fathi et al Virology 97–105 [1986]).

The construct to be inserted can be linear or circular. A circular DNAis preferred, especially a plasmid. The construct contains sequencesflanking the left and the right side of a naturally occurring deletion,e.g. deletion II, within the MVA genome (Altenburger, W., Suter, C. P.and Altenburger J. (1989) Arch. Virol. 105, 15–27). The foreign DNAsequence is inserted between the sequences flanking the naturallyoccurring deletion.

For the expression of at least one nucleic acid, it is necessary forregulatory sequences, which are required for the transcription of thenucleic acid to be present upstream of the nucleic acid. Such regulatorysequences are known to those skilled in the art, and includes forexample those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

The construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al Virol. 52, 456–467 [1973; Wigler et al Cell 777–785 [1979]by means of electroporation (Neumann et al EMBO J. 1, 841–845 [1982]),by microinjection (Graessmann et al Meth. Enzymology 101, 482–492(1983)), by means of liposomes (Straubinger et al Methods in Enzymology101, 512–527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl.Acad. Sci. USA 77, 2163–2167 (1980)) or by other methods known to thoseskilled in the art. Transfection by means of liposomes is preferred.

The recombinant priming and boosting vectors of the present inventioncan have a tropism for a specific cell type in the mammal. By way ofexample, the recombinant vectors of the present invention can beengineered to infect professional APCs such as dendritic cells andmacrophages. Dendritic cells are known to be orchestrators of asuccessful immune response especially that of a cell mediated response.It has been shown that ex vivo treatment of dendritic cells with antigenor viral vectors containing such a target antigen, will induceefficacious immune responses when infused into syngeneic animals orhumans (see Nestle F O, et al. Vaccination of melanoma patients withpeptide- or tumor lysate-pulsed dendritic cells, Nat Med. 1998March;4(3):328–32 and Kim C J, et al. Dendritic cells infected withpoxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro. JImmunother. 1997 July;20(4):276–86. The recombinant vectors can alsoinfect tumour cells. Alternatively, the recombinant vectors are able toinfect any cell in the mammal.

Other examples of vectors include ex vivo delivery systems, whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection andcompacted DNA-mediated transfection.

The vector may be a plasmid DNA vector. As used herein, “plasmid” refersto discrete elements that are used to introduce heterologous DNA intocells for either expression or replication thereof. Selection and use ofsuch vehicles are well within the skill of the artisan. Many plasmidsare available, and selection of appropriate plasmid will depend on theintended use of the plasmid, i.e. whether it is to be used for DNAamplification or for DNA expression, the size of the DNA to be insertedinto the plasmid, and the host cell to be transformed with the plasmid.Each plasmid contains various components depending on its function(amplification of DNA or expression of DNA) and the host cell for whichit is compatible. The plasmid components generally include, but are notlimited to, one or more of the following: an origin of replication, oneor more marker genes, an enhancer element, a promoter, a transcriptiontermination sequence and a signal sequence.

Both expression and cloning plasmids generally contain nucleic acidsequence that enable the plasmid to replicate in one or more selectedhost cells. Typically in cloning plasmids, this sequence is one thatenables the plasmid to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2m plasmid origin issuitable for yeast, and various viral origins (e.g. SV 40, polyoma,adenovirus) are useful for cloning plasmids in mammalian cells.Generally, the origin of replication component is not needed formammalian expression plasmids unless these are used in mammalian cellscompetent for high level DNA replication, such as COS cells.

Most expression plasmids are shuttle plasmids, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another class of organisms for expression. For example, a plasmidis cloned in E. Coli and then the same plasmid is transfected into yeastor mammalian cells even though it is not capable of replicatingindependently of the host cell chromosome.

Advantageously, an expression and cloning plasmid may contain aselection gene also referred to as selectable marker. This gene encodesa protein necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with theplasmid containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene canbe used which facilitates the selection for transformants due to thephenotypic expression of the marker gene. Suitable markers for yeastare, for example, those conferring resistance to antibiotics G418,hygromycin or bleomycin, or provide for prototrophy in an auxotrophicyeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.

Since the replication of plasmids is conveniently done in E. Coli, an E.Coli genetic marker and an E. Coli origin of replication areadvantageously included. These can be obtained from E. Coli plasmids,such as pBR322, Bluescript© plasmid or a pUC plasmid, e.g. pUC18 orpUC19, which contain both E. Coli replication origin and E. Coli geneticmarker conferring resistance to antibiotics, such as ampicillin.

Suitable selectable markers for mammalian cells are those that enablethe identification of cells which have taken up 5T4 nucleic acid, suchas dihydrofolate reductase (DHFR, methotrexate resistance), thymidinekinase, or genes conferring resistance to G418 or hygromycin. Themammalian cell transformants are placed under selection pressure whichonly those transformants which have taken up and are expressing themarker are uniquely adapted to survive. In the case of a DHFR orglutamine synthase (GS) marker, selection pressure can be imposed byculturing the transformants under conditions in which the pressure isprogressively increased, thereby leading to amplification (at itschromosomal integration site) of both the selection gene and the linkedDNA that encodes 5T4. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth,together with closely associated genes which may encode a desiredprotein, are reiterated in tandem within the chromosomes of recombinantcells. Increased quantities of desired protein are usually synthesisedfrom thus amplified DNA.

Expression and cloning plasmids usually contain a promoter that isrecognised by the host organism and is operably linked to 5T4 nucleicacid. Such a promoter may be inducible or constitutive. The promotersare operably linked to DNA encoding 5T4 by removing the promoter fromthe source DNA by restriction enzyme digestion and inserting theisolated promoter sequence into the plasmid. Both the native 5T4promoter sequence and many heterologous promoters may be used to directamplification and/or expression of 5T4 DNA. The term “operably linked”refers to a juxtaposition wherein the components described are in arelationship permitting them to function in their intended manner. Acontrol sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example,the α-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Their nucleotide sequences have been published, therebyenabling the skilled worker operably to ligate them to DNA encoding 5T4,using linkers or adaptors to supply any required restriction sites.Promoters for use in bacterial systems will also generally contain aShine-Delgarno sequence operably linked to the DNA encoding 5T4.

Preferred expression plasmids are bacterial expression plasmids whichcomprise a promoter of a bacteriophage such as phagex or T7 which iscapable of functioning in the bacteria. In one of the most widely usedexpression systems, the nucleic acid encoding the fusion protein may betranscribed from the plasmid by T7 RNA polymerase (Studier et al,Methods in Enzymol. 185; 60–89, 1990). In the E. Coli BL21(DE3) hoststrain, used in conjunction with pET plasmids, the T7 RNA polymerase isproduced from the λ-lysogen DE3 in the host bacterium, and itsexpression is under the control of the IPTG inducible lac UV5 promoter.This system has been employed successfully for over-production of manyproteins. Alternatively the polymerase gene may be introduced on alambda phage by infection with an int-phage such as the CE6 phage whichis commercially available (Novagen, Madison, USA). other plasmidsinclude plasmids containing the lambda PL promoter such as PLEX(Invitrogen, NL), plasmids containing the trc promoters such aspTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE), orplasmids containing the tac promoter such as pKK223-3 (PharmaciaBiotech) or PMAL (new England Biolabs, Mass., USA).

Moreover, the 5T4 gene according to the invention preferably includes asecretion sequence in order to facilitate secretion of the polypeptidefrom bacterial hosts, such that it will be produced as a soluble nativepeptide rather than in an inclusion body.

The peptide may be recovered from the bacterial periplasmic space, orthe culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulatedor constitutive and are preferably derived from a highly expressed yeastgene, especially a Saccharomyces cerevisiae gene. Thus, the promoter ofthe TRP1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene,a promoter of the yeast mating pheromone genes coding for the a- orα-factor or a promoter derived from a gene encoding a glycolytic enzymesuch as the promoter of the enolase, glyceraldehyde-3-phosphatedehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphateisomerase, phosphoglucose isomerase or glucokinase genes, the S.cerevisiae GAL 4 gene, the S. pombe nmt 1 gene or a promoter from theTATA binding protein (TBP) gene can be used. Furthermore, it is possibleto use hybrid promoters comprising upstream activation sequences (UAS)of one yeast gene and downstream promoter elements including afunctional TATA box of another yeast gene, for example a hybrid promoterincluding the UAS(s) of the yeast PH05 gene and downstream promoterelements including a functional TATA box of the yeast GAP gene (PH05-GAPhybrid promoter). A suitable constitutive PHO5 promoter is e.g. ashortened acid phosphatase PH05 promoter devoid of the upstreamregulatory elements (UAS) such as the PH05 (-173) promoter elementstarting at nucleotide-173 and ending at nucleotide-9 of the PH05 gene.

5T4 gene transcription from plasmids in mammalian hosts may becontrolled by promoters derived from the genomes of viruses such aspolyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, aviansarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40(SV40), from heterologous mammalian promoters such as the actin promoteror a very strong promoter, e.g. a ribosomal protein promoter, and fromthe promoter normally associated with 5T4 sequence, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding 5T4 by higher eukaryotes may beincreased by inserting an enhancer sequence into the plasmid. Enhancersare relatively orientation and position independent. Many enhancersequences are known from mammalian genes (e.g. elastase and globin).However, typically one will employ an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100–270) and the CMV early promoter enhancer. Theenhancer may be spliced into the plasmid at a position 5′ or 3′ to 5T4DNA, but is preferably located at a site 5′ from the promoter.

Advantageously, a eukaryotic expression plasmid encoding 5T4 maycomprise a locus control region (LCR). LCRs are capable of directinghigh-level integration site independent expression of transgenesintegrated into host cell chromatin, which is of importance especiallywhere the 5T4 gene is to be expressed in the context of apermanently-transfected eukaryotic cell line in which chromosomalintegration of the plasmid has occurred, in plasmids designed for genetherapy applications or in transgenic animals.

Eukaryotic expression plasmids will also contain sequences necessary forthe termination of transcription and for stabilising the mRNA. Suchsequences are commonly available from the 5′ and 3′ untranslated regionsof eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding 5T4.

An expression plasmid includes any plasmid capable of expressing 5T4nucleic acids that are operatively linked with regulatory sequences,such as promoter regions, that are capable of expression of such DNAs.Thus, an expression plasmid refers to a recombinant DNA or RNAconstruct, such as a plasmid, a phage, recombinant virus or otherplasmid, that upon introduction into an appropriate host cell, resultsin expression of the cloned DNA. Appropriate expression plasmids arewell known to those with ordinary skill in the art and include thosethat are replicable in eukaryotic and/or prokaryotic cells and thosethat remain episomal or those which integrate into the host cell genome.For example, DNAs encoding 5T4 may be inserted into a plasmid suitablefor expression of cDNAs in mammalian cells, e.g. a CMV enhancer-basedplasmid such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

Particularly useful for practising the present invention are expressionplasmids that provide for the transient expression of DNA encoding 5T4in mammalian cells. Transient expression usually involves the use of anexpression plasmid that is able to replicate efficiently in a host cell,such that the host cell accumulates many copies of the expressionplasmid, and, in turn, synthesises high levels of 5T4. For the purposesof the present invention, transient expression systems are useful e.g.for identifying 5T4 mutants, to identify potential phosphorylationsites, or to characterise functional domains of the protein.

Construction of plasmids according to the invention employs conventionalligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired. If desired, analysis to confirm correct sequences in theconstructed plasmids is performed in a known fashion. Suitable methodsfor constructing expression plasmids, preparing in vitro transcripts,introducing DNA into host cells, and performing analyses for assessing5T4 expression and function are known to those skilled in the art. Genepresence, amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA, dot blotting (DNA orRNA analysis), or in situ hybridisation, using an appropriately labelledprobe which may be based on a sequence provided herein. Those skilled inthe art will readily envisage how these methods may be modified, ifdesired.

5T4 Antigen, Fragments and Variants

5T4 antigen, as referred to herein, includes peptides and otherfragments of 5T4 which retain at least one common antigenic determinantof 5T4.

“Common antigenic determinant” means that the derivative in question atleast one antigenic function of 5T4. Antigenic functions includespossession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against a naturally occurring ordenatured 5T4 polypeptide or fragment thereof, or the ability to bindHLA molecules and induce a 5T4-specific immune response. Thus 5T4 asprovided by the present invention includes splice variants encoded bymRNA generated by alternative splicing of a primary transcript, aminoacid mutants, glycosylation variants and other covalent derivatives of5T4 which retain the physiological and/or physical properties of 5T4.Exemplary derivatives include molecules wherein the protein of theinvention is covalently modified by substitution, chemical, enzymatic,or other appropriate means with a moiety other than a naturallyoccurring amino acid. Such a moiety may be a detectable moiety such asan enzyme or a radioisotope. Further included are naturally occurringvariants of 5T4 found with a particular species, preferably a mammal.Such a variant may be encoded by a related gene of the same gene family,by an allelic variant of a particular gene, or represent an alternativesplicing variant of the 5T4 gene.

Derivatives which retain common antigenic determinants can be fragmentsof 5T4. Fragments of 5T4 comprise individual domains thereof, as well assmaller polypeptides derived from the domains. Preferably, smallerpolypeptides derived from 5T4 according to the invention define a singleepitope which is characteristic of 5T4. Fragments may in theory bealmost any size, as long as they retain one characteristic of 5T4.Preferably, fragments will be between 5 and 400 amino acids in length.Longer fragments are regarded as truncations of the full-length 5T4 andgenerally encompassed by the term “5T4”. Advantageously, fragments arerelatively small peptides of the order of 5 to 25 amino acids in length.Preferred are peptides about 9 amino acids in length.

Derivatives of 5T4 also comprise mutants thereof, which may containamino acid deletions, additions or substitutions, subject to therequirement to maintain at least one feature characteristic of 5T4.Thus, conservative amino acid substitutions may be made substantiallywithout altering the nature of 5T4, as may truncations from the 5′ or 3′ends. Deletions and substitutions may moreover be made to the fragmentsof 5T4 comprised by the invention. 5T4 mutants may be produced from aDNA encoding 5T4 which has been subjected to in vitro mutagenesisresulting e.g. in an addition, exchange and/or deletion of one or moreamino acids. For example, substitutional, deletional or insertionalvariants of 5T4 can be prepared by recombinant methods and screened forimmuno-crossreactivity with the native forms of 5T4.

Moreover, variant peptides can be screened for superior HLA bindingcapabilities using the program “Peptide Binding Predictions” devised byK. Parker at the National Institutes of Health (see Parker, K. C et al.1994.J.Immunol. 152:163).

The fragments, mutants and other derivative of 5T4 preferably retainsubstantial homology with 5T4. As used herein, “homology” means that thetwo entities share sufficient characteristics for the skilled person todetermine that they are similar in origin and function. Preferably,homology is used to refer to sequence identity. Thus, the derivatives of5T4 preferably retain substantial sequence identity with the sequence ofSEQ ID No. 2.

“Substantial homology”, where homology indicates sequence identity,means more than 40% sequence identity, preferably more than 45% sequenceidentity and most preferably a sequence identity of 50% or more, asjudged by direct sequence alignment and comparison.

Sequence homology (or identity) may moreover be determined using anysuitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andare advantageously set to the defined default parameters.

Advantageously, “substantial homology” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (seehttp://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.The BLAST programs were tailored for sequence similarity searching, forexample to identify homologues to a query sequence. The programs are notgenerally useful for motif-style searching. For a discussion of basicissues in similarity searching of sequence databases, see Altschul etal. (1994) Nature Genetics 6:119–129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov performthe following tasks:

blastp compares an amino acid query sequence against a protein sequencedatabase;

blastn compares a nucleotide query sequence against a nucleotidesequence database;

blastx compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase;

tblastn compares a protein query sequence against a nucleotide sequencedatabase dynamically translated in all six reading frames (bothstrands).

tblastx compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM Display a histogram of scores for each search; default is yes.(See parameter H in the BL/.ST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page). See also EXPECT andCUTOFF.

ALIGNMENTS Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).The valid alternative choices include: PAM40, PAM120, PAM250 andIDENTITY. No alternate scoring matrices are available for BLASTN;specifying the MATRIX directive in BLASTN requests returns an errorresponse.

STRAND Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149–163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Clayerie & States (1993) Computers and Chemistry17:191–201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g., hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g., “XXXXXXXXX”).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.

Alternatively, sequence homology may be determined by algorithms such asFastA, available at http://biology.ncsa.uiuc.edu/BW30/BW.cgi. FastA isconsidered to be superior to BLAST for alignment of short sequences.Advantageously, the FastA algorithm is employed using default parametersat http://biology.ncsa.uiuc.edu/BW30/BW.cgi.

Preferably, the protein or derivative thereof of the invention isprovided in isolated form. “Isolated” means that the protein orderivative has been identified and is free of one or more components ofits natural environment. Isolated 5T4 includes 5T4 in a recombinant cellculture. 5T4 present in an organism expressing a recombinant 5T4 gene,whether the 5T4 protein is “isolated” or otherwise, is included withinthe scope of the present invention.

In the vaccination of humans against tumours, the use of non-human TAAsis preferred. The invention accordingly provides canine 5T4. Canine 5T4is advantageously provided for use as a vaccine component in humans, inorder to elicit an immune response to human 5T4 in a human subject.

The sequence of canine 5T4 is set forth in SEQ. ID. No. 3. Sequencesfrom other canine sources are obtainable by those skilled in the art,for example by hybridisation with a nucleic acid probe derived from SEQ.ID. No. 3.

Exemplary nucleic acids can thus be characterised as those nucleotidesequences which encode a canine 5T4 protein and hybridise to the DNAsequences set forth SEQ ID No. 3, or a selected fragment of said DNAsequence. Preferred are such sequences encoding canine 5T4 whichhybridise under high-stringency conditions to the sequence of SEQ ID No.3.

Stringency of hybridisation refers to conditions under which polynucleicacid hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general, the stability of a hybrid is afunction of sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na+ at 65–68° C. High stringency conditions can beprovided, for example, by hybridisation in an aqueous solutioncontaining 6×SSC, 5× Denhardt's, 1% SDS (sodium dodecyl sulphate), 0.1Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as nonspecific competitor. Following hybridisation, high stringency washingmay be done in several steps, with a final wash (about 30 min) at thehybridisation temperature in 0.2–0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridisation inthe above described solution but at about 60–62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove described solution at about 50–52° C. In that case, the final washis performed at the hybridisation temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds.(1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).Optimal hybridisation conditions have to be determined empirically, asthe length and the GC content of the probe also play a role.

Advantageously, the invention moreover provides nucleic acid sequencewhich are capable of hybridising, under stringent conditions, to afragment of SEQ. ID. No. 3. Preferably, the fragment is between 15 and50 bases in length. Advantageously, it is about 25 bases in length.

Given the guidance provided herein, the nucleic acids of the inventionare obtainable according to methods well known in the art. For example,a DNA of the invention is obtainable by chemical synthesis, usingpolymerase chain reaction (PCR) or by screening a genomic library or asuitable cDNA library prepared from a source believed to possess canine5T4 and to express it at a detectable level.

Chemical methods for synthesis of a nucleic acid of interest are knownin the art and include triester, phosphite, phosphoramidite andH-phosphonate methods, PCR and other autoprimer methods as well asoligonucleotide synthesis on solid supports. These methods may be usedif the entire nucleic acid sequence of the nucleic acid is known, or thesequence of the nucleic acid complementary to the coding strand isavailable. Alternatively, if the target amino acid sequence is known,one may infer potential nucleic acid sequences using known and preferredcoding residues for each amino acid residue.

An alternative means to isolate the gene encoding canine 5T4 is to usePCR technology as described e.g. in section 14 of Sambrook et al., 1989.This method requires the use of oligonucleotide probes that willhybridise to canine 5T4 nucleic acid. Strategies for selection ofoligonucleotides are described below.

Libraries are screened with probes or analytical tools designed toidentify the gene of interest or the protein encoded by it. For cDNAexpression libraries suitable means include monoclonal or polyclonalantibodies that recognise and specifically bind to canine 5T4;oligonucleotides of about 20 to 80 bases in length that encode known orsuspected canine 5T4 cDNA from the same or different species; and/orcomplementary or homologous cDNAs or fragments thereof that encode thesame or a hybridising gene. Appropriate probes for screening genomic DNAlibraries include, but are not limited to oligonucleotides, cDNAs orfragments thereof that encode the same or hybridising DNA; and/orhomologous genomic DNAs or fragments thereof.

A nucleic acid encoding canine 5T4 may be isolated by screening suitablecDNA or genomic libraries under suitable hybridisation conditions with aprobe, i.e. a nucleic acid disclosed herein including oligonucleotidesderivable from the sequences set forth in SEQ ID NO. 3. Suitablelibraries are commercially available or can be prepared e.g. from celllines, tissue samples, and the like.

As used herein, a probe is e.g. a single-stranded DNA or RNA that has asequence of nucleotides that includes between 10 and 50, preferablybetween 15 and 30 and most preferably at least about 20 contiguous basesthat are the same as (or the complement of) an equivalent or greaternumber of contiguous bases set forth in SEQ ID No. 3. The nucleic acidsequences selected as probes should be of sufficient length andsufficiently unambiguous so that false positive results are minimised.The nucleotide sequences are usually based on conserved or highlyhomologous nucleotide sequences or regions of canine 5T4. The nucleicacids used as probes may be degenerate at one or more positions. The useof degenerate oligonucleotides may be of particular importance where alibrary is screened from a species in which preferential codon usage inthat species is not known.

Preferred regions from which to construct probes include 5′ and/or 3′coding sequences, sequences predicted to encode ligand binding sites,and the like. For example, either the full-length cDNA clone disclosedherein or fragments thereof can be used as probes. Preferably, nucleicacid probes of the invention are labelled with suitable label means forready detection upon hybridisation. For example, a suitable label meansis a radiolabel. The preferred method of labelling a DNA fragment is byincorporating α32P dATP with the Klenow fragment of DNA polymerase in arandom priming reaction, as is well known in the art. Oligonucleotidesare usually end-labelled with γ32P-labelled ATP and polynucleotidekinase. However, other methods (e.g. non-radioactive) may also be usedto label the fragment or oligonucleotide, including e.g. enzymelabelling, fluorescent labelling with suitable fluorophores andbiotinylation.

After screening the library, e.g. with a portion of DNA includingsubstantially the entire canine 5T4-encoding sequence or a suitableoligonucleotide based on a portion of said DNA, positive clones areidentified by detecting a hybridisation signal; the identified clonesare characterised by restriction enzyme mapping and/or DNA sequenceanalysis, and then examined, e.g. by comparison with the sequences setforth herein, to ascertain whether they include DNA encoding a completecanine 5T4 (i.e., if they include translation initiation and terminationcodons). If the selected clones are incomplete, they may be used torescreen the same or a different library to obtain overlapping clones.If the library is genomic, then the overlapping clones may include exonsand introns. If the library is a cDNA library, then the overlappingclones will include an open reading frame. In both instances, completeclones may be identified by comparison with the DNAs and deduced aminoacid sequences provided herein.

It is envisaged that the nucleic acid of the invention can be readilymodified by nucleotide substitution, nucleotide deletion, nucleotideinsertion or inversion of a nucleotide stretch, and any combinationthereof. Such mutants can be used e.g. to produce a canine 5T4 mutantthat has an amino acid sequence differing from the canine 5T4 sequencesas found in nature. Mutagenesis may be predetermined (site-specific) orrandom. A mutation which is not a silent mutation must not placesequences out of reading frames and preferably will not createcomplementary regions that could hybridise to produce secondary mRNAstructure such as loops or hairpins.

The foregoing considerations may also be applied to the isolation ofalternative murine (SEQ. ID. No. 2) or human (SEQ. ID. No. 1) 5T4antigens.

Administration of Vectors Encoding 5T4

A pharmaceutical composition according to the invention is a compositionof matter comprising a vector encoding a 5T4 antigen, as described, asan active ingredient. The active ingredients of a pharmaceuticalcomposition comprising the active ingredient according to the inventionare contemplated to exhibit excellent therapeutic and/or prophylacticactivity, for example, in the treatment and/or prophylaxis of tumours orother diseases associated with cell proliferation, infections andinflammatory conditions, when administered in amount which depends onthe particular case. Dosage regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound may be administered in a convenient manner such asby the oral, intravenous (where water soluble), intramuscular,subcutaneous, intranasal, intradermal or suppository routes orimplanting (e.g. using slow release molecules). Depending on the routeof administration, the active ingredient may be required to be coated ina material to protect said ingredients from the action of enzymes, acidsand other natural conditions which may inactivate said ingredient.

In order to administer the active compound by other than parenteraladministration, it will be coated by, or administered with, a materialto prevent its inactivation. For example, the active compound may beadministered in an adjuvant, co-administered with enzyme inhibitors orin liposomes. Adjuvant is used in its broadest sense and includes anyimmune stimulating compound such as interferon. Adjuvants contemplatedherein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin.

Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes.

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene gloycol, and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of superfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the sterilised active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze-drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

When the active compound is suitably protected as described above, itmay be orally administered, for example, with an inert diluent or withan assimilable edible carrier, or it may be enclosed in hard or softshell gelatin capsules, or it may be compressed into tablets, or it maybe incorporated directly with the food of the diet. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. The amountof active compound in such therapeutically useful compositions in suchthat a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac, sugar or both. A syrup orelixir may contain the active compound, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavouring such ascherry or orange flavour. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such as active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredients are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

Regimes for administration of 5T4-expressing vectors according to thepresent invention may be determined by conventional efficacy testing.Especially preferred, however, are regimes which include successivepriming and boosting steps. It is observed that such regimes achievesuperior breaking of immune tolerance and induction of CTL responses. Ina preferred embodiment, the priming step is undertaken using a non-viralvector, such as a plasmid encoding 5T4, whilst boosting is undertakenusing a viral vector, such as a poxvirus vector, encoding 5T4 (seeSchneider et al., 1998 Nat Med 4:397–402).

Administration of 5T4 Antigen

In general, approaches outlined above relating to the administration of5T4-encoding nucleic acids may be used for the administration of 5T4antigen, as a conventional vaccine preparation, for the therapy and/orprophylaxis of tumours.

In general, vaccines may be prepared 5T4 antigen. The preparation ofvaccines which contain an 5T4 as active ingredient(s) is known to oneskilled in the art. Typically, such vaccines are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation may also be emulsified, or theprotein encapsulated in liposomes. The active immunogenic ingredientsare often mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further examples of adjuvants and other agents include aluminiumhydroxide, aluminium phosphate, aluminium potassium sulphate (alum),beryllium sulphate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.).

Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel(aluminium hydroxide), or a mixture of Amphigen and Alhydrogel are used.Only aluminium hydroxide is approved for human use.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminium hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulatedto contain a final concentration of immunogen in the range of from 0.2to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose.

5T4 may be formulated into the vaccine as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids such as acetic, oxalic, tartaric and maleic. Saltsformed with the free carboxyl groups may also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine and procaine.

Measurement of the Efficacy of 5T4 Administration

5T4 activity as an immunotherapeutic molecule may be assessed accordingto any techniques known in the art, including assays for antibodyproduction, induction of CTL responses and tumour regression models.Exemplary techniques are set forth in the following examples.

The invention is further described, for the purposes of illustrationonly, in the following examples in which reference is made to thefollowing Figures.

FIG. 1 a shows a gene construct;

FIG. 1 b shows a gene construct;

FIG. 2 a shows a photographic representation;

FIG. 2 b shows a photographic representation;

FIG. 3 a present a graph;

FIG. 3 b present a graph;

FIG. 4 a presents a graph;

FIG. 4 b presents a graph;

FIG. 4 c presents a graph;

FIG. 5 presents a graph;

FIG. 6 presents a graph;

FIG. 7 presents a graph;

FIG. 8 presents a graph; and

FIG. 9 presents a graph;

In slightly more detail:

FIG. 1 a is a map of recombinant vaccinia virus MVA. Transgenes areplaced under the control of the vaccinia virus synthetic early/latepromoter. The Lac Z gene is under control of the vaccinia virus 7.5kearly/late promoter. The DNA regions flanking these genes are derivedfrom the deleted region two of MVA, thus allowing homologousrecombination into this site.

FIG. 1 b is a map of the recombinant vaccinia virus WR. The transgenesare under the control of the vaccinia virus synthetic early/latepromoter. The Lac Z gene is under control of the vaccinia virus 7.5kearly/late promoter. The DNA regions flanking the genes are derived fromthe thymidine kinase (tk) gene of Wyeth strain VV, thus allowingrecombination into this site.

FIG. 2 shows a western blot of recombinant vaccinia virus expressinghuman 5T4. Samples are run on a 12% SDS PAGE and transferred to anitrocellulose membrane.

FIG. 2 a: The blot is probed with a 1:500 dilution of MAb 5T4(anti-human 5T4). Bound antibody is visualised with a anti-mouse HRPconjugated antibody and ECL. Lane 1: recombinant WR clone 1 expressinghuman 5T4; Lane 2: recombinant WR clone 2 expressing human 5T4; Lane 3:BS-C-1 cells infected with WR; Lane 4: uninfected BS-C-1 cells; Lane 5:B16 melanoma cells; Lane 6: B16 cell line expressing human 5T4.

FIG. 2 b: The blot is probed with a 1:500 dilution of rabbit anti-mouse5T4. Lane 1: recombinant WR expressing LacZ; Lane 2: recombinant WRexpressing mouse 5T4; lane 3: recombinant WR expressing human 5T4; lane4: recombinant MVA expressing mouse 5T4.

FIGS. 3 a and 3 b are graphs which show that inoculation of mice withMVA-h5T4 Protects against Challenge with CT26 expressing h5T4

FIG. 4 a is a graph which shows that inoculation with MVA-h5T4 inducesanti-tumour activity against B16 tumours expressing h5T4.

FIGS. 4 b and 4 c are graphs which show that inoculation with MVA-m5T4induces anti-tumour activity against B16 tumours expressing m5T4.

FIG. 5 is a graph which shows that MVA-h5T4 induces tumour therapy inmice with pre-established lung tumours.

FIG. 6 is a graph which shows that mice that are vaccinated withMVA-m5T4 develop tumours at a slower rate than those that receive thecontrol vaccine.

FIG. 7 is a graph which shows that mice that were vaccinated withMVA-m5T4 had a lower tumour burden than those mice that receivedMVA-LacZ treatment.

EXAMPLES Example 1

Construction of Recombinant Poxvirus Vectors

Propagation of Vaccinia Virus

The highly attenuated strain MVA is derived from the replicationcompetent strain Ankara and has endured over 570 passages in primarychick embryo fibroblast cells. MVA replication was initially thought tobe restricted to CEF cells as only minimal replication in mammaliancells was reported. However, further analysis has shown that BabyHamster Kidney cells (BHK-21) are able to support high titre productionof MVA. MVA may thus be grown on BHK-21 or primary CEF cells (Carroll &Moss (1997) Virology 238:198–211).

To prepare CEF cells, 10 day old chick embryos are gutted and limbs andhead are removed before being minced and trypsinised in a solution of0.25% trypsin and incubation at 37° C. The cell suspension is filteredthrough a course filter mesh and cells are washed and concentrated bycentrifugation at 2000 rpm in a Sorvall RC-3B at 1500 rpm for 5 mins.Cells are suspended in MEM containing 10% FCS, aliquotted into 175 cmflasks and incubated at 37° C. in a CO₂ incubator. When monolayers are95% confluent they are trypsinised and used to seed additional flasks orsix well plates. Alternatively, primary cultures are transferred to a31° C. incubator for later use (Sutter and Moss (1992) Proc Natl AcadSci USA 89:10847–10851).

Preparation of Crude, Semi-Purified and Purified Virus Stocks

Crude virus stocks are prepared for initial recombinant virus analysisor as viral stocks used for subsequent high titre virus preparations.Vaccinia virus preparations can be semi-purified by centrifuging outcell membranes and nuclei or by additional steps involving sucrosecentrifugation to prevent contamination by pre-expressed recombinantprotein products and cellular organelles. Methods used are amodification of those described by Earl et al., in: Ausubel et al.(Eds.), (1991) Current Protocols in Molecular Biology, pp.16.16.1–16.16.17, New York: Greene Publishing Associates and WileyInterscience; Earl and Moss, ibid, pp. 16.17.1–16.17.16; Earl and Moss,ibid, pp. 16.18.1–16.18.10; and Bronte et al., (1997) Proc Natl Acad SciUSA 94(7):3183–3188.

Crude Virus

MVA is grown in either CEF or BHK-21 (obtained from the ATCC) and WR isgrown in HeLa or BS-C-1 (ATCC) in 175 cm² tissue culture flasks.Briefly, confluent monolayers are infected with an moi of approx. 1 pfuwith MVA or WR. Virus is suspended in 10 ml MEM containing 2% FCS andadded to 175 cm² flasks containing confluent cell monolayers. Afterinoculation for 1 hour at 37° C. an additional 20 ml MEM containing 2%FCS is added. After 48–72 hours infected cells are scraped into themedium and pelleted at 1500 g for 5 mins. For crude virus preparationscells are resuspended 2 ml MEM (2%) per 175 cm² flask. Cells are freezethawed three times, sonicated and aliquotted into 1 ml freezing tubes. Arepresentative aliquot is freeze thawed and titred to determine virusconcentration. Virus stocks are stored below −20° C.

Semi-Pure Preparations

Infected cells are harvested as described previously (Earl et al.; Earland Moss; 1991). After centrifugation cells are resuspended in PBS (2ml/175 cm² flask) and homogenised by 30–40 strokes in a tight fittingglass dounce homogeniser, on ice. Cell breakage is checked bymicroscopy. Nuclei, cellular organelles and membranes are removed by acentrifugation at 300 g for 5 mins (4° C.), keep supernatant. The cellpellet is resuspended in 1 ml/175 cm² flask and centrifugation repeated.The supernatants are pooled, aliquoted and stored.

Purified Preparation

Infected cells are harvested as previously described (Earl et al.; Earland Moss; 1991) and resuspended in 10 mM Tris.Cl, pH 9.0 (2 ml/flask),keeping samples on ice from this point of the procedure. Homogenise asdescribed previously using 10 mM Tris. The lysate is sonicated (on ice)using an XL 2015 sonicating cup (Misonics, USA) at maximum output (500W) for 1 min. The sample is placed on ice for 1 min and the sonicationrepeated up to 3 times. A maximum of 5 ml is sonicated at a time, andice is replenished during sonication. The lysate is gently layered ontoa cushion of 17 ml of 36% sucrose (in 10 mM Tris.Cl, pH 9.0) in a SW-27centrifuge tube. Lyates are centrifuged for 80 mins in an SW-27 rotor at13 500 rpm (32,900×g), 4° C. The supernatant is discarded and the viralpellet resuspended in sterile PBS and sonicated in a cup sonicator for 1min (on ice). Concentrated virus is aliquoted and stored at below −20°C.

Example 2

Construction and Characterisation of Recombinant Virus VectorsExpressing 5T4

Murine and human 5T4 genes are cloned into WR (pSC65) (Chakrabarti etal., (1997) Biotechniques 23:1094–7) and MVA (pLW22) transfer plasmidsto allow homologous recombination into targeted regions of therespective viral genomes.

Recombinant MVA and WR Expressing Human and Murine 5T4

The 1.4 kb murine and human 5T4 (supplied by P. Stern Paterson InstituteManchester) genes are excised from pBSII-m5T4 (pBluescript (Stratagene)containing the 5T4 cDNA) and pBSII-h5T4 (Myers et al., (1994) JBC269:9319–9324) respectively by Eco RI and Bam HI restriction digestion.The fragments are blunt ended by “filling in” with dNTPs and DNApolymerase. The blunt ended fragments are cloned into the PmeI site ofpLW22 (an MVA transfer plasmid, consisting of an early late promoter(Chakrabarti et al., 1997) upstream of an MCS. Adjacent is a VV 7.5 KbLacZ cassette, for detection of recombinant virus; see FIG. 1 a), andSma I site of pSC65 (b) (Chakrabarti et al., 1997). pLW22 and pSC65direct homologous recombination to deletion region II and the tk generespectively.

In constructs destined for vaccination of human subjects, the LacZ geneunder the control of the 7.5k promoter is omitted. Recombinant plaquesare identified by live immunostaining using an anti-5T4 monoclonalantibody, as described previously (Wyatt et al., (1996) Vaccine14:1451–1458).

Wild type MVA, supplied by B. Moss (NIH, Bethesda, USA) is grown in CEFcells from a plaque purified clone and is the same isolate that was usedto make previously described recombinant viruses (Sutter and Moss (1992)Proc Natl Acad Sci USA 89:10847–10851, Sutter et al (1994) Vaccine12:1032–1040, Hirsch et al (1996) J Virol 70:3741–3752, Carroll and Moss(1995) Biotechniques 19: 352–355, Wyatt et al (1995) Virology210:202–205, Sutter et al., (1995) FEBS Lett. 371:9–12, Wyatt et al.(1996) Vaccine 14, 1451–1458, Carroll et al (1997) Vaccine, 15:387–394,Carroll and Moss (1997) Virology 238:198–211) The WR stock is suppliedby B. Moss (NIH), from the ATCC isolate (see e.g. Earl and Moss, 1991).The WR stock is prepared in HeLa S3 cells (ATCC).

The protocol used to make recombinant MVA virus is similar to thatdescribed previously (Carroll & Moss (1997) Virology 238:198–211).Briefly: BHK-21 or CEF cells are infected at an moi of 0.1 with an MVAstock. Plasmid DNA is diluted to 2 μg in 100 ul d.H₂O and mixed with 30μg lipofectin (BRL) diluted to 100 μl with sterile d.H2O. After 10minutes incubation at RT the Lipofectin/DNA solution is added toinfected cells overlaid with Opti MEM. Five hours after incubation at37° C. cells media is aspirated and replaced with MEM containing 2% FCS.Cells are harvested after a further 36 hours incubation and assayed forthe expression of P-gal on CEF or BHK-21 cells in the presence of5-bromo-3-indolyl-D-galactosidase. Isolated plaques are plaque purifiedat least an additional 3 times. After plaque purification small viralstocks are prepared in CEF or BHK-21 cells.

Protocols for the construction of recombinant WR are similar to thosedescribed previously (Carroll and Moss (1995) Biotechniques 19: 352–355;Earl and Moss, 1991). Briefly: recombination is carried out as for MVA.However, BS-C-1 cells are used and recombinant plaques assayed in 143Btk⁻ cells in the presence of BrdU with an agar overlay containing thesubstrate for the LacZ gene, 5-bromo-3-indolyl-D-galactosidase. Neutralred is used to detect LAC Z negative spontaneous tk⁻ virus forevaluation of virus homogeneity.

Recombinant protein expression is initially analysed by direct plaqueimmunostaining using antibodies specific for h5T4 and m5T4 using amethod similar to that described previously (Carroll & Moss (1997)Virology 238:198–211). Briefly: recombinant viruses are plaqued onmonolayers of BS-C-1 cells, fixed with acetone/methanol and treated withMAb 5T4 (Hole N, and Stern P L, (1990) Int J Cancer 45(1):179–184).Anti-mouse HRP conjugated antibody and dianizidine substrate are used tovisualise recombinant 5T4 protein expression. 5T4 expressed protein isfurther characterised by western blotting under non-reducing conditions,as the MAb recognises a conformational epitope. As can be seen in FIG. 2recombinant viruses express high levels of protein at the appropriatesize of 72 kDa. The stock is checked for homogeneity bydouble-immunostaining as described in Carroll & Moss (1997) Virology238:198–211.

Example 3

Animal Models to Illustrate Immunological Cross Protection of Mouse 5T4with Human 5T4.

To determine if the 5T4 gene product from one species can induceimmunity to 5T4 in another species, the recombinant poxviruses aretested in murine tumour models. The mouse models are based on CT26, achemically induced adenocarcinoma of BALB/c origin (Brittain et al.,(1980) Cancer Res. 40:179–184), and on B16, a melanoma line derived fromC57 B6 mice. Both the CT26 line and B16 are stably transformed toexpress human and murine 5T4. Mice are injected I.V. (to induce lungnodules, CT26) or subcutaneously (CT26 and B16) to make single masssubcutaneous tumours.

Groups of 7 BALB/c mice were inoculated three times IV or IM with 1×10⁷pfu of MVA-h5T4 (here the 5T4 antigen is called OBA1) construct on days0, 21 and 42. Mice were then challenged IV with 5×10⁵ tumour cells thatwere stably transfected with human 5T4. 14 days after challenge mouselungs were removed and lung nodules counted.

Results 3

The results shown in FIGS. 3 a and 3 b demonstrate that mice vaccinatedwith MVA-h5T4 showed anti-tumour activity when challenged with thesyngeneic tumour line CT26 expressing the h5T4 protein. Mann-Whitneystatistical analysis show that protection after vaccination withMVA-h5T4 is significant, compared to vaccination with MVA-LacZ or PBS(p<0.05).

Example 4

Groups of 5 C57 BL 6 mice were inoculated twice at a three week intervalwith 10⁷ pfu of MVA-h5T4 (IV or IM) or MVA-Lac Z (IV). Mice werechallenged with 5×10⁵ B16-h5T4 cells. Tumours sizes were recorded at 2day intervals post tumour challenge. Individual tumour areas are given.

Groups of 5 and 7 C57 BL 6 mice were inoculated twice at a three weekinterval with 10⁷ pfu of MVA-m5T4 (IV or IM) or MVA-Lac Z (IV). Micewere challenged with 5×10⁵ B16-m5T4 cells. Tumours sizes were recordedat 2 day intervals post tumour challenge. Individual tumour areas aregiven.

Results 4

FIG. 4 a shows that vaccination with MVA-h5T4 clearly has an anti-tumoureffect when mice are challenged with B16-h5T4. Mann-Whitney statisticalanalysis of data in FIG. 4 a demonstrates that tumour retardation aftervaccination with MVA-h5T4 is significant, compared to vaccination withMVA-LacZ (p<0.05).

FIGS. 4 b and 4 c show that vaccination with MVA-m5T4 clearly has ananti-tumour effect when mice are challenged with B16-m5T4. Mann-Whitneystatistical analysis of data in FIG. 4 c demonstrates that tumourretardation after vaccination with MVA-m5T4 is significant, compared tovaccination with MVA-LacZ (p<0.05).

Example 5

Female BALB/c mice were injected IV with 5×10⁵ CT26-h5T4 cells. After 3days macro lung tumours establish. Mice were treated on day 3 and 10post tumour inoculation with 10⁷ pfu of MVA-Lac Z, MVA-h5T4 (groups of10 mice) or PBS (group of 5 mice). Lungs are stained and tumours counted14 days post tumour inoculation.

Results 5

It is clear from FIG. 5 that treatment with MVA-h5T4 has a significanttherapeutic effect on established CT26 lung nodules expressing h5T4.Statistical analysis (Mann-Whitney) shows that therapy with MVA-h5T4 issignificant when compared to MVA-Lac Z or PBS (p<0.05).

Example 6

CT26-m5T4 and B16-m5T4 Self Antigen Model

C57 BL6 mice are inoculated I.V. twice at a three week interval with1×10⁷ pfu of either MVA-LacZ (n=3) or MVA-m5T4 (n=6). Three weeks afterthe last vaccination mice are challenged S.C. with 5×10⁵ B16 expressingm5T4. Development of sub cutaneous (S.C.) tumours is monitored.

FIG. 6 shows that mice that are vaccinated with MVA-m5T4 develop tumoursat a slower rate than those that receive the control vaccine.Additionally, the tumours in the m5T4 vaccinated mice are on average 5fold smaller in volume (10 fold smaller by day 13) compared to thosemice that receive the MVA-LacZ treatment. This protective property canbe paralleled by the m5T4 antibody response induced in these mice.

Example 7

BALB/c mice are inoculated I.V. twice at a three week interval with1×10⁷ pfu of either MVA-LacZ (n=5) or MVA-m5T4 (n=6). Three weeks afterthe last vaccination mice are challenged with 5×10⁵ CT26 expressingm5T4. 12 days after challenge mouse lungs are removed and tumour nodulescounted in a blinded manner. Results 7 FIG. 7 shows that mice that werevaccinated with MVA-m5T4 had a lower tumour burden than those mice thatreceived the MVA-LacZ treatment. This protective property can beparalleled by the m5T4 antibody response induced in these mice.

Example 8

Induction of 5T4 Antibody Responses in C57 and BALB/c Mice

Mice are vaccinated as above with MVA-m5T4 and MVA-h5T4, and bled 10days after each vaccination.

Results 8

MVA-m5T4 is able to overcome tolerance in both BALB/c and C57 B1 6 mice,after two inoculations. Additionally, mice primed with DNA followed byMVA do not show signs of an antibody response to m5T4.

It is therefore shown, in two murine tumour models, that vaccinationwith MVA expressing murine 5T4 has protective properties againstsyngeneic tumour cells expressing m5T4. These anti-tumour properties canbe paralleled to the anti-m5T4 immune response (measured by ELISA).Additionally, it is shown that the induction of such an immune responsedoes not induce auto immune toxicity in these animals.

Example 9

Immune Response to 5T4 Using Primer Boost Vaccination

In order to evaluate the efficacy of MVA and naked DNA vectors to induceimmunity to 5T4 in BALB/c and C57 BL6 mice, mice are inoculated usingsuccessive priming and boosting with both naked DNA and MVA vectorsencoding mouse and human 5T4. In more detail, mice are inoculated with1×10⁷ pfu of MVA-5T4 i.v., 50 g pCl-h5T4 (25 g/hind leg) or 25 gpCl-m5T4 (12.5 g/hind leg) on day 0, with a second (booster) inoculationon day 21 with MVA-5T4. On day 29 the mice are bled and antibody titresdetermined by ELISA.

Results 9

The following titres (defined as that dilution giving an OD above twicethe background OD of MVA-LacZ) as shown in Tables 2 and 3 are observed:

TABLE 2 Murine 5T4 Antibody Titres from Mice Inoculated with MVA and DNAvectors expressing human and murine 5T4 BALB/c 1:10000 <1:1000 <1:1000<1:1000 C57 BL6 1:16000 <1:1000 <1:1000 <1:1000

TABLE 3 Human 5T4 Antibody Titres from Mice Inoculated with MVA and DNAvectors expressing human and murine 5T4. BALB/c 1:5000 <1:1000 >1:32000 1:32000 C57 BL6 1:4000 <1:1000 >1:32000 >1:32000

The results show that use of either DNA:MVA prime:boost with aheterologous 5T4 antigen (h5T4) or an MVA:MVA prime:boost withheterologous or homologous 5T4 (H5T4 or m5T4) is effective in raising ahigh titre of antibodies.

Example 10

Modified Forms of 5T4 for Cancer Immunotherapy

It is possible to modify human 5T4 to enhance its immunogenicity andthus induce more efficacious immunotherapy responses. To do this,identification of HLA CTL epitopes and modification of such epitopes toimprove binding to the HLA molecule, and thus more efficient CTLinduction, is performed using the programme “Peptide BindingPredictions” devised by K. Parker at the National Institutes of Health;<http:www.bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform> (seeParker et al., J. Immunol., 152:163 (1994)). The following results areobtained for human (Table 4) and murine (Table 5) 5T4 9mers:

TABLE 4 Human 5T4 9mers (SEQ ID NOS 5–17, respectively in order ofappearance) binding to HLA A 0201 Rank Start Sequence Dissociation Time1 97 FLTGNQLAV (SEQ ID NO:5) 319.939 2 364 ALIGAIFLL (SEQ ID NO:6)284.974 3 351 SLQTSYVFL (SEQ ID NO:7) 176.240 4 368 AIFLLVLYL (SEQ IDNO:8) 137.482 5 283 GLPHIRVFL (SEQ ID NO:9) 117.493 6 358 FLGIVLALI (SEQID NO:10) 110.379 7 81 NLTEVPTDL (SEQ ID NO:11) 87.586 8 95 NLFLTGNQL(SEQ ID NO:12) 79.041 9 222 FLYLPRDVL (SEQ ID NO:13) 63.174 10 373VLYLNRKGI (SEQ ID NO:14) 56.754 11 365 LIGAIFLLV (SEQ ID NO:15) 30.89012 290 FLDNNPWVC (SEQ ID NO:16) 28.109 13 301 HMADMVTWL (SEQ ID NO:17)27.207

TABLE 5 Murine 5T4 9mers (SEQ ID NOS 18–27, respectively in order ofappearance) binding to human HLA A 0201 Rank Start Sequence DissociationTime 1 307 YMADMVAWL (SEQ ID NO:18) 3680.892 2 81 NLLEVPADL (SEQ IDNO:19) 324.068 3 97 FLTGNQMTV (SEQ ID NO:20) 319.939 4 370 ALIGAIFLL(SEQ ID NO:21) 284.974 5 228 FLFLPRDLL (SEQ ID NO:22) 178.158 6 357SLQTSYVFL (SEQ ID NO:23) 176.240 7 374 AIFLLVLYL (SEQ ID NO:24) 137.4828 289 GLAHVKVFL (SEQ ID NO:25) 117.493 9 364 FLGIVLALI (SEQ ID NO:26)110.379 10 379 VLYLNRKG (SEQ ID NO:27) 56.754

Example 11

Mutation of H5T4 to Improve Binding of HLA A0201

The above data derived from the Parker Peptide Binding PredictionsProgramme indicates that mutation of the human AA sequence starting atposition 301 from YMADMVAWL (SEQ ID NO:18) when changed to HMADMVTWL(SEQ ID NO:17) leads to a 10 fold increase in half time of dissociationto HLA A0201.

Results 11

Additionally, mutations in the h5T4 9mer starting at 81 from NLTEVPTDL(SEQ ID NO:11) to NLLEVPADL (SEQ ID NO:19) lead to a 4 fold increase inthe dissociation half time of the 9mer to HLA A 0201.

Example 12

Toxicity Studies

Absence of Autoimmune Toxicity

The purpose of this study was to study the possible effects of inducingautoimmue toxicity against 5T4 in the murine model

Experimental Design

Groups of 5 BALB/c and C57 BL6 mice were inoculated IV with 10⁷ pfu ofrecombinant vaccinia virus MVA expressing human (MVA-h5T4) and murine5T4 (MVA-m5T4). As a negative control mice were inoculated with MVAexpressing E. Coli LacZ (MVA-LacZ) or PBS.

Female BALB/c mice were inoculated a total of four times over an14-month period. C57 BL6 mice were inoculated 3 times over an 14 monthperiod. Blood samples were taken after inoculation and evaluated for 5T4specific antibody by ELISA.

Results 12a

Antibody response: After 2 inoculations mice inoculated with MVA-m5T4and MVA-h5T4 had high levels of anti-m5T4 and anti-h5T4 respectively, intheir serum (see Table 2 and Table 3).

Toxicity: Mice were observed for signs of ill health on a daily basis.At no time during the past 14 months did the physical appearance ofanimals inoculated with MVA-m5T4 or MVA-h5T4 differ from those animalsinoculated with MVA-Lac Z or PBS. Two reports on the health of theanimals, prepared by a qualified veterinarian, were prepared that statesall animals appear healthy.

Summary

Groups of BALB/c and C57 BL6 mice were inoculated up to four times overan 14 month period with MVA expressing m5T4 (MVA-m5T4) or MVA expressingh5T4 (MVA-h5T4) antigens and checked for signs of toxicity. Though micewere shown to have high titres of antibodies to m5T4 there were no signsof ill health over the 14-month period. A qualified veterinarian hasassessed animals and found them to show no signs of ill health,indicating an absence of auto immune toxicity.

Thus, inoculation of BALB/c or C57 BL6 mice with MVA-h5T4 or MVA-m5T4induces an antibody response to h5T4 and m5T4 respectively. Such aresponse has no detrimental effect on the health of the mice.

Example 12b

Effects of 5T4 Auto-Immunity on Fertility

5T4 is found on human and murine placenta. Accordingly, is possible thatan anti mouse 5T4 immune response may prevent mice becoming pregnant oreffect the health of the foetus. We carried out extensive studies toaddress these issues. The purpose of this study was to assess theeffects of an immune response in both BALB/c and C57 BL6 female mice tom5T4 on pregnancy.

Experimental Design

Groups of 5 female BALB/c and C57 BL6 mice were inoculated on threeoccasions IV with 10⁷ pfu of MVA-m5T4, MVA-LacZ or PBS. At specifictimes following the final inoculations (day 10, 30 and 60) mice weremated and evaluated for their ability to become pregnant and give birthto healthy pups.

Results 12b

(i) C57 BL6 Mouse Study

TABLE 6 10 DAY Study MVA-m5T4 × 3 MVA-LacZ × 3 PBS No. of pregnancies4/5 = 80% 4/5 = 80% 3/5 = 60% No. Live births 24 30 18 Average littersize  6  7.5  6

TABLE 7 30 DAY Study MVA-m5T4 × 3 No. of pregnancies 4/5 = 80% No. Livebirths 22 Average litter size  5.5

TABLE 8 60 DAY Study MVA-m5T4 × 3 No. of pregnancies 5/5 = 100% No. Livebirths 33 Average litter size  6.6(ii) BALB-c Mouse Study

TABLE 9 10 DAY Study MVA-m5T4 × 3 MVA-LacZ × 3 PBS No. of pregnancies5/5 = 100% 4/5 = 80% 4/5 = 80% No. Live births 30 23 22 Average littersize  6  5.75  5.5 No. surviving to 24 = 80.0% 19 = 82.6% 22 = 100%weaning Ratio F:M 13:11 14:5 11:11 Average weights 10.4 g 11.2 g 10.1 g

TABLE 10 45 DAY Study MVA-m5T4 × 3 No. of pregnancies 4/4 = 100% No.Live births 24 Average litter size  6 No. surviving to 24 = 100.0%weaning Ratio F:M 15:9 Average weights 11.4 gSummary

BALB/c and C57 BL6 mice were injected with an MVA recombinant virusexpressing m5T4 and an anti-m5T4 antibody response was induced. Thesemice were mated and were shown to get pregnant and give birth to pups atthe same rate as mice that were vaccinated with a control virus (seetables 6–10) Additionally, there was no effect on the health of the pupsto weaning. Thus, inoculation with MVA-m5T4 and induction of m5T4antibody response does not have a detrimental effect on (i) Fertility ofBALB/c and C57 BL6 mice; (ii) Number of live births and (iii) Weight andsurvival of pups to weaning.

Example 12c

Distribution of 5T4 in Normal Human Tissues

The purpose of this study was to carry out an independent evaluation of5T4 distribution in normal human tissues. Some past studies havesuggested that normal tissue expressing 5T4 could potentially be atarget for an immune response induced against this tumour antigen eventhough the level of 5T4 expression in normal tissue associated withsmall vessels was found to be over 1000-fold lower than that associatedwith placenta. It was not clear whether the staining was specific orreflected some cross reactivity by the MAb.

Experimental Design

Slide preparations of thirty two different tissue types from 3 differentdonors were evaluated by a qualified pathologist under GLP conditions.Cryosections of each tissue sample were stained with three differentconcentrations of Mab specific for 5T4.

Results 12c

The summary Table 11 indicates that tissue sections from all essentialorgans including: brain, CNS, liver and kidney were negative for 5T4expression. Some weak staining in one or more of the donor tissues wasevident in several non-essential tissues. It should be noted that someof the positive staining was observed in tissues derived fromindividuals that had died from cancer.

Although past studies using immunohistochemical analysis have revealedthat some 5T4 staining was observed in “some small vessels” of somenon-cancerous organs which “appeared to be weakly staining” and include;Kidney (glomeruli), bladder (epithelium), small intestine (villousepithelium), uterus (endometril glands), cervix (endocervical glands)and skin (basal epidermis), this independent study shows that 5T4 is notexpressed on cells of essential organs, furthermore, expression in somenormal tissues is at a low level and is sporadic as it is rarely seen inall three donor samples. Thus, this data indicates that 5T4 tissueexpression is more stringently restricted compared to some other tumourantigens that have been employed in tumour immunotherapy trials.Therefore these findings reiterate the view that 5T4 is an excellentcandidate antigen for cancer immunotherapy.

TABLE 11 Summary of 5T4 Tissue Distribution ¹Donors Tissue 1 2 3Staining Distribution Adrenal − − − Bladder +/++ − − Urothelium onlystained (Nt and 1:8) Blood Cells Bone Marrow Breast − − − BrainCerebellum − − − Brain Cortex − − − Colon − − − Endothelium − + −Variable staining in endothelium Fallopian Tube + − − Variable stainingof epithelium Heart − − − Kidney − − − Liver − − − Uterus Cervix − − −²Uterus Endometrium − − + Epithelial cells positive Lung − − − LymphNode − − − Ovary + − − Mesothelial and epithelial staining Pancreas − −− Parathyroid − − − Pituitary + − + Individual cells staining Placenta+++ +++ +++ All staining on trophoblasts surface Prostate − − − Skin − −− Spinal Cord − − − Spleen − − − Striated Muscle − − − Testis − − −Thymus Thyroid − − − ²Ureter + + + Urothelium only stained (Nt and 1:8)Gastric Antrum − − − Gastric Body − − − Ileum − − − ²Duodenum ++/+ − +Muscularis mucosa only stained Eye Cornea − − − Eye Lens − − − EyeRetina − − − ¹5T4 staining intensity was defined by visual analysisrelative to placenta trophoblasts (+++) and negative control staining(−). ²Tissues derived from cancer patients

Example 13

ScFv Fusion Protein In Vivo Anti Tumour Efficacy Data

The purpose of the study was to test the efficacy of a series of singlechain antibody fusion proteins.

Experimental Design

CT26 Cells Expressing Human 5T4 (CT26-h5T4) and CT26-neo

Cells were pre-incubated with:

PBS, LscFv-1, LscFv-2, B7-scFv, ScFv-Ig

LscFv-1 and 2 were expressed in a BHK cell line. LscFv-1 was purifiedvia its Histidine tag on a Nickel column and scFv-2 was purified using afiltration system. B7-scFv was purified from a BHK line via a His tagand scFv-Ig was purified via a filtration column. The concentration ofeach scFv used in the experiment was defined as the amount of proteinrequired to saturate binding of CT26-h5T4 cells in a FACS assay.

CT26-h5T4 and CT26-neo cells were pre-incubated with saturating amountsof each scFv and incubated for 1 hour. After washing cells 5×105 cellswere injected subcutaneously into the flanks of syngeneic BALB/c mice.

Tumour measurements were taken every two days and the volume calculated.

Results 13

FIG. 8: CT26-neo

There is not a significant difference between the groups accept in thecase of LscFv-1, for which appears to be 3-fold reduction in tumour sizecompared to the PBS control 36 days after tumour inoculation.

FIG. 9: CT26-h5T4

Tumours treated with all the scFv constructs had a significant effect ontumour growth. 4 of the 5 mice treated with scFv-1 were tumour free onday 36. On day 36 scFv-1 treated tumour were >60 fold smaller thantumours treated with PBS.

When a similar experiment was carried out using a mouse melanoma line(B16) engineered to express h5T4 there was no anti-tumour effect.

Summary

In summary there appears to be no benefit of fusing B7 or IgG to the 5T4specific scFv in the CT26 and B16 murine models. The scFv alone may bemore efficacious due to its higher binding affinity (as shown in BIACOREcompared to B7-scFV). Thefore this data indicates that the scFv alonehas a significant effect on tumour retardation and immune enhancingmolecules fused to the scFv may not be required to show an effect ontumour retardation in the 5T4 model.

1. A method for eliciting an anti-tumor immunotherapeutic response to atumor in a subject, comprising immunizing the subject with a mammalian5T4 antigen, wherein the 5T4 antigen induces an anti-tumorimmunotherapeutic response to a tumor in the subject.
 2. The methodaccording to claim 1, wherein the 5T4 antigen is modified to differ froma naturally occurring mammalian 5T4 antigen and comprises an HLA CTLpeptide epitope of a mammalian 5T4 antigen, and wherein said anti-tumorimmunotherapeutic response comprises a CTL response.
 3. The methodaccording to claim 1, wherein said immunizing is with a non-human,mammalian 5T4 antigen as an immunizing agent.
 4. The method according toclaim 3, wherein said agent further comprises one or more adjuvants. 5.The method according to claim 3, wherein the non-human, mammalian 5T4antigen is modified to differ from a naturally occurring mammalian 5T4antigen, and comprises a peptide epitope of a mammalian 5T4 antigen, andwherein the modified 5T4 antigen induces a CTL response.
 6. The methodaccording to claim 1, wherein the 5T4 antigen is modified to differ froma naturally occurring mammalian 5T4 antigen.
 7. The method according toclaim 1, wherein said immunizing is with a murine 5T4 antigen.
 8. Themethod according to claim 1, wherein said immunizing is with a canine5T4 antigen.
 9. The method of claim 1, wherein said immunizing is with ahuman 5T4 antigen.
 10. The method according to claim 1, wherein theanti-tumor immunotherapeutic response comprises an antibody response.11. The method according to claim 1, wherein said immunizing is with a5T4 antigen modified to differ from a naturally occurring mammalian 5T4antigen and a pharmaceutically acceptable carrier, wherein the modified5T4 antigen comprises an HLA CTL peptide epitope of a mammalian 5T4antigen.
 12. The method according to claim 11, wherein said naturallyoccurring mammalian 5T4 antigen is a human 5T4 antigen.